Functional annotation of human long noncoding RNAs via molecular phenotyping

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.Peer reviewe

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1.

Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors

Loss of Tbk1 kinase activity protects mice from diet-induced metabolic dysfunction

Objective: TANK Binding Kinase 1 (TBK1) has been implicated in the regulation of metabolism through studies with the drug amlexanox, an inhibitor of the IκB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since amlexanox is a dual IKKε/TBK1 inhibitor, the specific metabolic contribution of TBK1 is not clear. Methods: To distinguish metabolic functions unique to TBK1, we examined the metabolic profile of global Tbk1 mutant mice challenged with an obesogenic diet and investigated potential mechanisms for the improved metabolic phenotype. Results and conclusion: We report that systemic loss of TBK1 kinase function has an overall protective effect on metabolic readouts in mice on an obesogenic diet, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor. Keywords: TBK1, IKKε, Obesity, Insulin, Insulin resistance, Metabolis

<i>Pab1-1</i> Is Not a General Suppressor of DNA Repair and Replication Stress Defects.

<p><i>A</i> and <i>B</i>, dilution series of the indicated strains were spotted onto YES plates, with or without HU at the indicated concentrations and temperatures.</p

Pab1 Dosage Is Inversely Correlated with Growth of <i>slx8-29</i> Cells.

<p><i>A</i>, <i>B and D</i>, dilution series of the indicated strains were spotted onto plates, with or without HU at the indicated concentrations and temperatures. The plates in <i>A</i> were YES also containing 5 μg/ml of anhydrotetracycline (ahTet), in <i>B</i> were minimum medium (EMM-LUAH) without thiamine (-B1) to induce the expression of GFP-Pab1 from the nmt41 promoter, and in <i>D</i> were YES without or with 3.5 mM HU. <i>C</i>, FLAG-IP of FLAG-tagged wild type Pab1 (lane 2, 5) or mutant Pab1-1 (lane 3, 6). Cells expressing untagged Pab1 (lane 1, 4) were used as control. 1% input and 20% of the FLAG-IP were analyzed by Western blotting with mouse-anti-FLAG, rabbit-anti-Ppa2 (Sunrise Science Products), and IRDye-conjugated anti-mouse⁶⁸⁰, anti-rabbit⁸⁰⁰ (Licor).</p

Impact of <i>pab1-1</i> on SUMO Pathway Activity.

<p><i>A</i>, Western blots of sumoylated proteins or tubulin of indicated strains grown at 25°C to mid log phase then shifted to 35°C for 3 h. <i>B</i>, Western blots of sumoylated proteins or tubulin of indicated strains. Ectopic expression of Pab1 under the <i>nmt41</i> or <i>nmt1</i> promoter was induced by growing cells in the absence of thiamine (–B1) for 48 h at 25°C. <i>C</i>, Western blots of Pli1-TAP, detected by peroxidase anti-peroxidase (<i>PAP</i>), or tubulin of indicated strains grown at 25°C to log phase.</p

<i>Rzr1-1</i> (Razor) a Suppressor of <i>slx8-29</i> (STUbL).

<p><i>A</i> and <i>D</i>, dilution series of the indicated strains were spotted onto YES plates, with or without drugs (HU: hydroxyurea; CPT: camptothecin) at the indicated concentrations and temperatures. <i>B</i>, three representative tetrad dissections are shown from a genetic cross between <i>rfp1Δ</i> and <i>rfp2Δ rzr1-1</i> cells. <i>C</i>, a schematic representation of the insertion mutation of <i>rzr1-1</i> in the conserved WD40 domain in the <i>pab1</i> locus. The indicated amino acids of the Bα subunits of PP2A in fission yeast (Pab1), human (B55) and budding yeast (Cdc55) are aligned, highlighting the high degree of sequence conservation across species. The "TEVI" insertion occurs at the end of a loop region between β3D and β4A in the seven-bladed β-propeller domain of Bα [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006165#pgen.1006165.ref024" target="_blank">24</a>].</p

List of yeast strains used in this study.

<p>List of yeast strains used in this study.</p

Impact of CDK Activity and the Cell Cycle Checkpoints on the <i>slx8-29</i> Phenotype.

<p><i>A</i>, differential interference contrast (DIC) light microscopy of the indicated strains. <i>Bar</i>, 5 μM. <i>B-D</i>, dilution series of the indicated strains were spotted onto YES plates, with or without drug.</p